Biol 568 Advanced Topics in Molecular Genetics

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Biol 568Advanced Topics in Molecular Genetics
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Chapter 9 Transcription

• Transcription in general
• RNA Polymerase
• Sigma factors
• Termination

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Fig 9.1 Overview of Transcription
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Overview of Transcription
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Basic Questions Regarding Transcription

• How does RNA Polymerase find promoters in DNA?
• How do regulatory proteins interact with RNA
  Polymerase and one another to regulate
  initiation, elongation termination of
  transcription?

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Transcription occurs by base pairing of unpaired
DNA

• Fig. 9.3

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Fig 9.4 General nature of transcription
Bases added through complementary base- pairing
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Fig 9.5 Transcription bubble
2 Turns unwound 18bp (12-20bp) RNA-DNA Hybrid
shorter than 12 bp maybe on 2-3bp
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Stages of Transcription

• Template recognition (binding)
• Three stages
• Initiation
• Elongation
• Termination

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Fig. 9.6 Three stages of transcription
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Bacterial RNA Pol structure
Fig 9.8 Nucleic acids are held in grooves in
RNA polymerase Size (bacterial) 90 x 95 x
160 Å (eukaryotic Pol are larger)
DNA
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Phosphodiester bond formation
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Phosphodiester bond formation

• RNA Pol reads the template 3 --gt 5
• RNA is synthesized 5 --gt 3
• Rate 40 nt / sec at 37C
• slower than DNA replication (800nt/sec)

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Chapter 9 Transcription

• Transcription in general
• RNA Polymerase
• Sigma factors
• Termination

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Fig 9.16 Eubacterial RNA Pol subunits
a2bbs
core a2bb 465,000 Dal
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Eubacterial RNA Pol Subunits

• a2bbs Holoenzyme
• a2bb Core
• bb Catalytic Center
• s Specific Promoter Recognition

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RNA Pol Functions

• Catalyzes RNA synthesis
• Supervises base pairing of substrate
  ribonucleotides with DNA
• Catalyzes formation of phosphodiester bonds

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Other RNA Pol of importance

• T3 and T7 RNA Polymerases
• single poolypeptide chain
• size lt100kDal
• Rate of syn 200 nt / sec at 37C
• Recognize only T3 and T7 phage promoters

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RNA Pol Binding

• Holoenzyme must bind
• Steps -
• Closed binary complex
• Open binary complex
• Ternary complex
• Synthesis begins

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Fig 9.19 RNA Pol binding
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Fig 9.19 RNA Pol binding
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Fig 9.20 RNA Pol bound to promoter
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Fig 9.20 RNA Pol bound to promoter
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Fig 9.11 RNA Pol bound to promoter
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RNA Pol in bacterial cell

• Free or bound holoenzyme in cell?
• Free or bound core enzyme in cell?
• Excess core is loosely bound as closed complexes
• 1/3 of RNA Pol is holoenzyme bound
  nonspecifically and in binary complexes
• 1/2 of RNA Pol is core enzyme engaged in txn

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Fig 9.12 RNA Pol distribution in cell
Little unbound enzyme
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How does RNA Pol find promoters?

• Bulk of DNA is not promoter regions
• promoter 60bp per gene
• E. coli genome 4.6 x 106 bp

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How does RNA Pol find promoters?

• Three models
• 1) Random diffusion to target
• 2) Random diffusion to any DNA followed by
  random displacement between DNA
• 3) Sliding along DNA

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Fig 9.23 Random diffusion to promoter
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Fig 9.24 Random displacement
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Fig 9.25 Sliding along DNA to promoter
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Promoter recognition

• sigma factor of holoenzyme
• consensus sequences in promoter
• start point of txn generally a purine

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Consensus Sequences

• -35 sequence
• T82T84G78A65C54A45
• -10 sequence
• T80A95T45A60A50T96
• Distance between -35 and -10
• 16 to 18 bp (range 15 to 20)
• sequence not important - spacing is
• UP element
• AT region further upstream present in some
  promoters
• Binding site for alpha subunits

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Fig 9.27 Typical promoter
Sigma factor interacts with these two consensus
sequences
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Analysis of proteins binding to DNA

• DNA footprinting
• locates specific DNA region bound by the protein
• limited DNase I digestion of labeled DNA with
  protein bound
• separation of fragments by gel electrophoresis

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Principle of DNA Footprinting
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Fig 9.29 DNA Footprinting
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Fig 9.29 Footprinting
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Fig 9.30 RNA Pol binding to promoter
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DNA Supercoiling

• TXn introduces positve and negative supercoiling
• In vitro
• TXn is initiated more efficiently when DNA is
  supercoiled
• The efficency of some promoters is influenced by
  degree of supercoiling
• Txn has a significant effect on the local DNA
  structure

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Fig 9.31 Supercoils generated in transcription
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Chapter 9 Transcription

• Transcription in general
• RNA Polymerase
• Sigma factors
• Termination

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Two modes of termination in bacteria

• Intrinsic terminators
• hairpin forms in RNA
• Rho dependent
• require action of rho protein

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Fig 9.27 Intrinsic Terminators
Hairpin forms in RNA GC rich stem Us at
end
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Rho dependent terminators

• Require the rho protein
• 46 kDal protein
• functions as a hexamer (275 kDal)
• Has an RNA binding domain ( C-rich region)
• Few in number in E. coli
• phage genes

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Fig 9.48 Rho-dependent terminator
50-90 bases in length rich in C, poor in G
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Fig 9.49 Model for Rho action
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Fig 9.49 Model for Rho action
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Chapter 9 Transcription

• Transcription in general
• RNA Polymerase
• Sigma factors
• Termination

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Alternative Sigma Factors

• E. coli sigma factors
• B. subtilis/phage SPO1 sigma factors

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Sigma Factors control Initiation

• Holoenzyme binds
• Sigma Factor interacts with -35,-10 regions
• Core enzyme only is needed for elongation

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Alternative Sigma Factors

• Substitution of sigma factors
• confers recognition of new promoters
• new -35,-10 regions interact with new sigma
  factor
• Expression of new sets of genes through
  substitution of sigma factors

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Alternative Sigma Factors

• Required in cases of changing environments -
  (nutrients, temperature)
• Different set of genes required for response

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Fig 9.33 E. coli Sigma Factors
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Alternative Sigma Factors

• In response to heat (stress)
• rpoH - switches on the heat shock response
• s32 - confers new specificity to core Pol
• s70 is replaced
• heat shock promoters recognized
• promoters of vegetative genes not recognized

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Alternative Sigma Factors

• specify new sets of genes for txn
• recognize new consensus sequences in promoters
  (-35, -10)
• Old sigma factor genes ---gt OFF
• New sigma factor genes ---gt ON
• Mutually exclusive transcription

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Alternative Sigma Factors

• Interaction with
• -35,-10 regions ---gt highly specific
• core polymerase --gt common mechanism

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Fig 9.36 Conserved regions of s70
2.1 2.2
2.3
(s only)
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Fig 9.37 s70 binding to -10 region
2.4 region a-helix
non-template strand
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Alternative s Factors in B.subtilis

• more widespread than in E. coli
• more than 10 alternative s factors known
• vegetative growth
• heat shock (stress)
• sporulation
• phage infection

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B. subtilis s factors

• vegetative s43 (E. coli s70)
• same holoenzyme structure
• a2bbs
• low amounts of other s factors

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B. subtilis s factors

• may be organized into cascades
• phage infection
• sporulation

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B. subtilis s factors

• one early gene product is a s factor
• specifies new set of genes (middle genes)
• a middle gene is a different s factor
• specifies new set of genes (late genes)

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Fig 9.41 Txn of phage SPO1
s70
s28
s33,34
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Phage SPO1 Transcription Cascade
s70
early
s28
middle
s33,34
late
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Sporulation s factor cascade

• vegetative cell to spore
• Similar to SPO1 phage infection
• cascade of s factors
• transcription of new sets of genes at each step

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Sporulation s factor cascade
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(No Transcript)
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Fig 9.44 s factor cascade in sporulation
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Fig 9.45 s factor cascade in sporulationCriss-
cross of regulation coordinates timing
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Chapter 9Transcription

• Transcription in general
• RNA Polymerase
• Sigma factors
• Termination

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Biol 568Advanced Topics in Molecular Genetics
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Chapter 10 The Operon

• Positive Negative Control
• Coordinate control of structural genes
• Repressors Inducers
• Specificity of Protein-DNA Interactions

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Trans and Cis-acting Elements

• Trans -acting element
• Any gene product that acts upon its target
• Cis -acting element
• Any sequence of DNA that as such acts upon the
  sequence to which it is physically linked

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Negative and Positive Regulation
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Fig 10.2 Coordinate control of structural genes
- Negative control
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Negative Control

• Classic mode of control in bacteria
• Repressor protein binds to Operator DNA sequence
  to block transcription
• Repressor - trans-acting factor
• Operator - cis-acting element

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Fig 10.1 Coordinate control of structural genes
- Positive control
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Positive Control

• Occurs in bacteria probably as frequently as
  negative regulation
• Most common in eukaryotes
• Proteins required for initiation of Txn
• Activators, transcription factors
• Trans-acting factors
• Binding sites
• Cis-acting elements

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Chapter 10 The Operon

• Positive Negative Control
• Coordinate control of structural genes
• Repressors Inducers
• Specificity of Protein-DNA Interactions

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Regulation of Operons

• Structural genes are coordinately controlled
• Coordinate regulation of genes in operon
• Single promoter/operator for all genes in operon

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Fig 10.3 Lac Operon

• Lac Operon
• One transcript
• Three proteins
• Controlled by same promoter/operator

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Fig 10.3 Lac Operon
b-galactosidase cleavage of lactose
(b-galactosides) Permease transports
b-galactosides into cell Transacetylase transfers
acetyl group from Acetyl CoA to b-galactosides
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Regulation of lac Operon

• Negative regulation
• lac repressor (lac I gene) binds with lac
  operator to block transcription
• Operon is transcribed unless repressor is bound
  to operator

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Fig 10.5 Repressor and RNA Pol binding sites
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Lac Repressor

• Size - 38kDal
• Functions as a tetramer
• 10 tetramers per cell (wild-type)
• constitutive expression of LacI gene

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Control of Repressor Activity

• If repressor is expressed constitutively, how is
  the operon turned on?
• How is the operon induced?

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Lac Operon as Model

• If no lactose in environment - no need for
  b-galactosidase
• When lactose is present, need to induce
  transcription of operon
• INDUCER molecule

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Chapter 10 The Operon

• Positive Negative Control
• Coordinate control of structural genes
• Repressors Inducers
• Specificity of Protein-DNA Interactions

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Fig 10.6 Lac Operon Induction
A
B
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Repressor Activity

• Modulated by small molecule effectors
• Two types
• Inducers
• result in production of proteins
• Co-repressors
• prevent production of proteins

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Modulating Repressor Activity

• Gratuitous Inducers
• cannot be metabolized
• IPTG - for lac operon
• (isopropyl thio-galactoside)

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Fig 10.7 Lac operon regulation
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Fig 10.8 Lac operon regulation
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Regulation of Operons

• In absence of inducer
• Operon is not transcribed
• In presence of inducer
• Operon is transcribed

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Regulation of Operons

• Repressors exhibit allosteric control
• one site of protein influences activity of
  another site
• Inducer binding alters repressors DNA binding
  ability

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Mutational Analysis

• Promoter and operator
• Targets for regulatory protein
• Cis-acting elements
• lacI locus
• Gene that codes for the repressor protein
• Trans-acting product

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Mutational Analysis

• Constitutive mutants
• expressed all of the time
• Uninducible mutants
• cannot be expressed

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Mutations in the Operator

• operator mutations are cis-acting
• control only adjacent lac genes
• oc mutation
• constitutive expression
• repressor cannot bind to operator

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Fig 10.9 Oc Mutation
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Oc Mutation

• Repressor protein cannot recognize DNA sequence
  of operator
• Cis-dominant mutation
• Controls adjacent genes
• No effect in other alleles!

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Lac I- Mutation

• Mutations in lac I repressor gene
• also constitutive expression

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Lac I- Mutation
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Other Lac I- Mutations

• lacI-d mutation
• constitutive expression
• repressor cannot bind to operator
• -d indicates it is dominant to wild-type
• mixed tetramer is non-functional
• trans-dominant or dominant negative

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Dominant Negatives

• Important tool in eukaryotic genetics
• A dominant negative protein
• Functions as part of a multimer

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Other Lac I- Mutations

• LacIs mutation
• Uninducible mutation
• repressor is unresponsive to inducer
• Lac Iq
• overexpression of repressor protein
• mutation in Lac I promoter

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Structure of operator

• Palindromic sequences
• Protected region
• Repressor contact
• Constitutive mutations

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Repressor Action

• constitutive expression
• binds with operator DNA sequence
• Inducer prevents binding with DNA

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Fig 10.14 Repressor-Inducer bindingTwo Models

Too slow
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Fig 10.15 Lac repressor structure
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Fig 10.16 Lac repressor structure
Dimer Tetramer
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Fig 10.16 Lac repressor structure
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Fig 10.18 Inducer changes repressor structure
NO Inducer
WITH Inducer
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Locations of mutations in lactose repressor

• Fig 10.19

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Chapter 10 The Operon

• Positive Negative Control
• Coordinate control of structural genes
• Repressors Inducers
• Specificity of Protein-DNA Interactions

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Repressor binds to operators
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Repressor binds to operators

• Weak Operators
• O2 410
• O3 - 83
• Deletions
• O2 or O3 repression reduced by 2-4X
• O2 O3 repression reduced by 100X !
• Binding to another operator ( other than O1) is
  important for repression

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Repressor binds to looped DNA
Fig 10.20
Fig 10.21
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Repressor interacts with RNA Pol

• RNA pol bound to Lac promoter
• RNA pol alone
• KB 1.9 X 107 M-1
• RNA pol in the presence of repressor
• KB 2.5 X 109 M-1
• When occupied by repressor the promoter is 100X
  more likely to be bound by RNA pol

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Repressor interacts with RNA Pol

• RNA stored at the promoter
• The RNA Pol-Repressor-DNA complex is blocked at
  the closed stage
• Transcription can begin immediately after
  induction

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Repressor is always bound to DNA

• Fig 10.22
• The equilibrium for repressor binding to random
  DNA

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Repressor is always bound to DNA

• The nonspecific equilibrium binding constant
• KA 2 106 M1.
• The concentration of nonspecific binding sites
• 4 106/7 103 M
• Free / Bound repressor 104
• All but 0.01 of repressor is bound to (random)
  DNA
• There are 10 molecules of repressor per cell
• There is no free repressor protein!

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Lac repressor binding
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Induction changes repressor distribution
Fig 10.24
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Chapter 10 The Operon

• Positive Negative Control
• Coordinate control of structural genes
• Repressors Inducers
• Specificity of Protein-DNA Interactions